Abstract: Clostridium difficile is responsible for more than 29,000 deaths in the US each year and the infection represents an urgent threat to public health worldwide. Of most concern is that the incidence of C. difficile infection (CDI) and disease severity is rapidly increasing in recent years due to the emergence of hypervirulent and antibiotic-resistant strains. CDI is mainly caused by two major toxins TcdA and TcdB, and toxin- neutralizing antibodies (antitoxins) are responsible for effective immunity. Therefore, it is important to measure host antitoxin responses since such information will be crucial for assisting physicians to predict CDI progression and recurrence, as well as to guide the use of antibody-based therapeutics. However, current assays to measure patient antitoxin responses are based on serum inhibition of toxins? biological activities on cultured cells; such assays are time consuming, laborious, and difficult to standardize. We aim to develop a serological ELISA for predicting protective immunity against primary and recurrent CDI in patients. This will help clinical management of CDI and also may be used as a companion diagnostic for passive and active immunotherapies. We have identified a broadly neutralizing antibody (bNAb) N3 and established a N3-based competition ELISA in that CDI patient sera were assayed for inhibiting the binding of biotinylated N3 to TcdB. We found that those sera with high neutralizing anti-TcdB titers are also significantly more potent in inhibiting N3 binding to TcdB than sera with low neutralizing titers. In this project, we will identify additional bNAbs from a panel of anti-TcdB neutralizing antibodies we have developed and evaluate the bNAbs-based ELISA in 240 banked serum samples from CDI patients. Our ultimate goal is to develop a simple serological ELISA screen for antitoxin protective immunity, and thus help the clinical management of CDI and assist the future use of passive and active immunotherapies against this debilitating disease.